The introduction of genetic material into a cell can facilitate expression of an encoded protein to complement a deficient or defective protein. The use of such technology allows for the treatment of disease as well as production of certain proteins in an in vitro application.
One method of introducing nucleic acids into a cell is mechanically, using direct microinjection. However this method is labor-intensive and, therefore, only practical for transfecting small numbers of cells such as eukaryotic germline cells for the production of transgenic systems. To be effective in treating a disease, a nucleic acid-based therapy typically must enter many cells.
Gene transfer entails distributing nucleic acids to target cells and then transferring the nucleic acid across a target cell membrane intact and, typically, into the nucleus in a form that can function in a therapeutic manner. In vivo gene transfer is complicated by serum interactions, immune clearance, toxicity and biodistribution, depending on the route of adminstration.
The in vivo gene transfer methods under study in the clinic consist almost entirely of viral vectors. Although viral vectors have the inherent ability to transport nucleic acids across cell membranes and some can integrate exogenous DNA into the chromosomes, they can carry only limited amounts of DNA and also pose risks. One such risk involves the random integration of viral genetic sequences into patient chromosomes, potentially damaging the genome and possibly inducing a malignant transformation. Another risk is that the viral vector may revert to a pathogenic genotype either through mutation or genetic exchange with a wild-type virus.
More recently, cationic lipids have been used to deliver nucleic acids to cells, allowing uptake and expression of foreign genes both in vivo and in vitro. While the mechanism by which cationic lipid carriers act to mediate transfection is not clearly understood, they are postulated to act in a number of ways with respect to both cellular uptake and intracellular trafficking. Some of the proposed mechanisms by which cationic lipids enhance transfection include: (i) compacting the DNA, protecting it from nuclease degradation and enhancing receptor-mediated uptake, (ii) improving association with negatively-charged cellular membranes by giving the complexes a positive charge, (iii) promoting fusion with endosomal membranes facilitating the release of complexes from endosomal compartments, and (iv) enhancing transport from the cytoplasm to the nucleus where DNA may be transcribed. When used for in vivo delivery, the role of the cationic lipid carriers is further complicated by the interactions between the lipid-nucleic acid complexes and host factors, e.g., the effects of the lipids on binding of blood proteins, clearance and/or destabilization of the complexes.
Typically, cationic lipids are mixed with a non-cationic lipid, usually a neutral lipid, and allowed to form stable liposomes, which liposomes are then mixed with the nucleic acid to be delivered. The liposomes may be large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs) or small unilamellar vesicles (SUVs). The liposomes are mixed with nucleic acid in solution, at concentrations and ratios optimized for the target cells to be transfected, to form cationic lipid-nucleic acid transfection complexes. Alterations in the lipid formulation allow preferential delivery of nucleic acids to particular tissues in vivo. PCT patent application numbers WO 96/40962, WO 96/40963. Certain preformed cationic liposome compositions are available, such as LIPOFECTIN.RTM. and LIPOFECTAMINE.RTM.. Another method of complex formation involves the formation of DNA complexes with mono- or poly-cationic lipids without the presence of a neutral lipid. These complexes are often not stable in water. Additionally, these complexes are adversely affected by serum (see, Behr, Acc. Chem. Res. 26:274-78 (1993)). An example of a commercially available poly-cationic lipid is TRANSFECTAM.RTM..
While the use of cationic lipid carriers for transfection is now well established, structure activity relationships are not well understood. It is postulated that different lipid carriers will affect each of the various steps in the transfection process (e.g., condensation, uptake, nuclease protection, endosomal release, nuclear trafficking, and decondensation) with greater or lesser efficiency, thereby making the overall transfection rate difficult to correlate with lipid structures. Thus, alterations in either the cationic or neutral lipid component do not have easily predictable effects on activity. For the most part, therefore, improvements to known cationic lipid-mediated delivery systems are dependent on empirical testing. When intended for in vivo transfection, new lipids and lipid formulations should be screened in vivo to accurately predict optimal lipids and formulations for transfection of target cells.
More recently, new cationic lipids have been prepared which exhibit excellent transfection properties when formulated with nucleic acids. See WO 95/14380, the disclosure of which is incorporated herein by reference. The compositions provided in WO 95/14380 are metabolizable in animal cells to components that are typically endogenous to the cells. Despite the properties associated with the novel cationic lipids, there exists a need for cationic lipids which are more hydrolytically stable and which can be formulated into suitable transfection compositions. The present invention provides such cationic lipids, along with methods for their preparation and use in lipid-based compositions.
Relevant Literature
Cationic lipid carriers have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., (1987) Proc. Natl. Acad. Sci. (USA), 84:7413-7416); mRNA (Malone et al., (1989) Proc. Natl. Acad. Sci. (USA) 86:6077-6081); and purified transcription factors (Debs et al., (1990) J. Biol. Chem. 265:10189-10192), in functional form. Literature describing the use of lipids as carriers for DNA include the following: Zhu et al., (1993) Science, 261:209-211; Vigneron et al., (1996) Proc. Natl. Acad. Sci. (USA), 93:9682-9686; Hofland et al., (1996) Proc. Natl. Acad. Sci. (USA), 93:7305-7309; Alton et al., (1993) Nat. Genet. 5:135-142; von der Leyen et al., (1995) Proc. Natl. Acad. Sci. (USA), 92:1137-1141; See also Stribling et al., (1992) Proc. Natl. Acad. Sci (USA) 89:11277-11281, which reports the use of lipids as carriers for aerosol gene delivery to the lungs of mice. For a review of liposomes in gene therapy, see Lasic and Templeton, (1996) Adv. Drug Deliv. Rev. 20:221-266.
The role of helper or neutral lipids in cationic lipid-mediated gene delivery is described in Felgner et al., (1994) J. Biol. Chem. 269(4): 2550-2561 (describing improved transfection using DOPE); and Hui et al., (1996) Biophys. J. 71: 590-599. The effect of cholesterol on liposomes in vivo is described in Semple et al., (1996) Biochem. 35(8): 2521-2525.